Sisario, D., Memmel, S., Doose, S., Neubauer, J., Zimmermann, H., Flentje, M., Djuzenova, C.S., Sauer, M., Sukhorukov, V.L.: Nanostructure of DNA repair foci revealed by superresolution microscopy.The FASEB Journal.fj.201701435-- (2018).
Induction of DNA double-strand breaks (DSBs) by ionizing radiation leads to formation of micrometer-sized DNA-repair foci, whose organization on the nanometer-scale remains unknown because of the diffraction limit (?200 nm) of conventional microscopy. Here, we applied diffraction-unlimited, direct stochastic optical-reconstruction microscopy (dSTORM) with a lateral resolution of ?20 nm to analyze the focal nanostructure of the DSB marker histone ?H2AX and the DNA-repair protein kinase (DNA-PK) in irradiated glioblastoma multiforme cells. Although standard confocal microscopy revealed substantial colocalization of immunostained ?H2AX and DNA-PK, in our dSTORM images, the 2 proteins showed very little (if any) colocalization despite their close spatial proximity. We also found that ?H2AX foci consisted of distinct circular subunits (?nanofoci?) with a diameter of ?45 nm, whereas DNA-PK displayed a diffuse, intrafocal distribution. We conclude that ?H2AX nanofoci represent the elementary, structural units of DSB repair foci, that is, individual ?H2AX-containing nucleosomes. dSTORM-based ?H2AX nanofoci counting and distance measurements between nanofoci provided quantitative information on the total amount of chromatin involved in DSB repair as well as on the number and longitudinal distribution of ?H2AX-containing nucleosomes in a chromatin fiber. We thus estimate that a single focus involves between ?0.6 and ?1.1 Mbp of chromatin, depending on radiation treatment. Because of their ability to unravel the nanostructure of DSB-repair foci, dSTORM and related single-molecule localization nanoscopy methods will likely emerge as powerful tools in biology and medicine to elucidate the effects of DNA damaging agents in cells.?Sisario, D., Memmel, S., Doose, S., Neubauer, J., Zimmermann, H., Flentje, M., Djuzenova, C. S., Sauer, M., Sukhorukov, V. L. Nanostructure of DNA repair foci revealed by superresolution microscopy.
Shirakashi, R., Yasui, T., Memmel, S., Sukhorukov, V.L.: Electro-microinjection of fish eggs with an immobile capillary electrode.Biomicrofluidics.9,064109 (2015).
Microinjection with ultra-fine glass capillaries is widely used to introduce cryoprotective agents and other foreign molecules into animal cells, oocytes, and embryos. The fragility of glass capillaries makes difficult the microinjection of fish eggs and embryos, which are usually protected by a hard outer shell, called the chorion. In this study, we introduce a new electromechanical approach, based on the electropiercing of fish eggs with a stationary needle electrode. The electropiercing setup consists of two asymmetric electrodes, including a μm-scaled nickel needle placed opposite to a mm-scaled planar counter-electrode. A fish egg is immersed in low-conductivity solution and positioned between the electrodes. Upon application of a short electric pulse of sufficient field strength, the chorion is electroporated and the egg is attracted to the needle electrode by positive dielectrophoresis. As a result, the hard chorion and the subjacent yolk membrane are impaled by the sharp electrode tip, thus providing direct access to the egg yolk plasma. Our experiments on early-stage medaka fish embryos showed the applicability of electro-microinjection to fish eggs measuring about 1 mm in diameter. We optimized the electropiercing of medaka eggs with respect to the field strength, pulse duration, and conductivity of bathing medium. We microscopically examined the injection of dye solution into egg yolk and the impact of electropiercing on embryos' viability and development. We also analyzed the mechanisms of electropiercing in comparison with the conventional mechanical microinjection. The new electropiercing method has a high potential for automation, e.g., via integration into microfluidic devices, which would allow a large-scale microinjection of fish eggs for a variety of applications in basic research and aquaculture.
Djuzenova, C.S., Fiedler, V., Memmel, S., Katzer, A., Hartmann, S., Krohne, G., Zimmermann, H., Scholz, C.-J., Polat, B., Flentje, M., Sukhorukov, V.L.: Actin cytoskeleton organization, cell surface modification and invasion rate of 5 glioblastoma cell lines differing in PTEN and p53 status.Experimental Cell Research.330,346 - 357 (2015).
Glioblastoma cells exhibit highly invasive behavior whose mechanisms are not yet fully understood. The present study explores the relationship between the invasion capacity of 5 glioblastoma cell lines differing in p53 and PTEN status, expression of mTOR and several other marker proteins involved in cell invasion, actin cytoskeleton organization and cell morphology. We found that two glioblastoma lines mutated in both p53 and PTEN genes (U373-MG and SNB19) exhibited the highest invasion rates through the Matrigel or collagen matrix. In DK-MG (p53wt/PTENwt) and GaMG (p53mut/PTENwt) cells, F-actin mainly occurred in the numerous stress fibers spanning the cytoplasm, whereas U87-MG (p53wt/PTENmut), U373-MG and SNB19 (both p53mut/PTENmut) cells preferentially expressed F-actin in filopodia and lamellipodia. Scanning electron microscopy confirmed the abundant filopodia and lamellipodia in the PTEN mutated cell lines. Interestingly, the gene profiling analysis revealed two clusters of cell lines, corresponding to the most (U373-MG and SNB19, i.e. p53 and PTEN mutated cells) and less invasive phenotypes. The results of this study might shed new light on the mechanisms of glioblastoma invasion.
Andronic, J., Shirakashi, R., Pickel, S.U., Westerling, K.M., Klein, T., Holm, T., Sauer, M., Sukhorukov, V.L.: Hypotonic Activation of the Myo-Inositol Transporter SLC5A3 in HEK293 Cells Probed by Cell Volumetry, Confocal and Super-Resolution Microscopy.PLoS ONE.10,e0119990 (2015).
Swelling-activated pathways for myo-inositol, one of the most abundant organic osmolytes in mammalian cells, have not yet been identified. The present study explores the SLC5A3 protein as a possible transporter of myo-inositol in hyponically swollen HEK293 cells. To address this issue, we examined the relationship between the hypotonicity-induced changes in plasma membrane permeability to myo-inositol Pino [m/s] and expression/localization of SLC5A3. Pino values were determined by cell volumetry over a wide tonicity range (100–275 mOsm) in myo-inositol-substituted solutions. While being negligible under mild hypotonicity (200–275 mOsm), Pino grew rapidly at osmolalities below 200 mOsm to reach a maximum of ~3 nm/s at 100–125 mOsm, as indicated by fast cell swelling due to myo-inositol influx. The increase in Pino resulted most likely from the hypotonicity-mediated incorporation of cytosolic SLC5A3 into the plasma membrane, as revealed by confocal fluorescence microscopy of cells expressing EGFP-tagged SLC5A3 and super-resolution imaging of immunostained SLC5A3 by direct stochastic optical reconstruction microscopy (dSTORM). dSTORM in hypotonic cells revealed a surface density of membrane-associated SLC5A3 proteins of 200–2000 localizations/μm2. Assuming SLC5A3 to be the major path for myo-inositol, a turnover rate of 80–800 myo-inositol molecules per second for a single transporter protein was estimated from combined volumetric and dSTORM data. Hypotonic stress also caused a significant upregulation of SLC5A3 gene expression as detected by semiquantitative RT-PCR and Western blot analysis. In summary, our data provide first evidence for swelling-mediated activation of SLC5A3 thus suggesting a functional role of this transporter in hypotonic volume regulation of mammalian cells.
Terpitz, U., Sukhorukov, V.L., Zimmermann, D.: Prototype for Automatable, Dielectrophoretically-Accessed Intracellular Membrane-Potential Measurements by Metal Electrodes.ASSAY and Drug Development Technologies.11,9-16 (2013).
Functional access to membrane proteins, for example, ion channels, of individual cells is an important prerequisite in drug discovery studies. The highly sophisticated patch-clamp method is widely used for electrogenic membrane proteins, but is demanding for the operator, and its automation remains challenging. The dielectrophoretically-accessed, intracellular membrane–potential measurement (DAIMM) method is a new technique showing high potential for automation of electrophysiological data recording in the whole-cell configuration. A cell suspension is brought between a mm-scaled planar electrode and a μm-scaled tip electrode, placed opposite to each other. Due to the asymmetric electrode configuration, the application of alternating electric fields (1–5 MHz) provokes a dielectrophoretic force acting on the target cell. As a consequence, the cell is accelerated and pierced by the tip electrode, hence functioning as the internal (working) electrode. We used the light-gated cation channel Channelrhodopsin-2 as a reporter protein expressed in HEK293 cells to characterize the DAIMM method in comparison with the patch-clamp technique.
Zimmermann, U., Bitter, R., Marchiori, P.E.R., Rüger, S., Ehrenberger, W., Sukhorukov, V.L., Schüttler, A., Ribeiro, R.V.: A non-invasive plant-based probe for continuous monitoring of water stress in real time: a new tool for irrigation scheduling and deeper insight into drought and salinity stress physiology.Theoretical and Experimental Plant Physiology.25,2 - 11 (2013).
The non-invasive, magnetic leaf patch clamp pressure probe (also termed ZIM-probe) allows for the first time to measure continuously turgor pressure changes of plant leaves over long periods of time with high precision and in real time. The probe has become an important tool in plant physiology, molecular biology and ecology, but also in agriculture because the probe is very robust and user-friendly. Growers receive the information about the water status of their plants by wireless telemetry, mobile network and internet on an as-needed basis and can thus adjust very precisely both the timing of irrigation and the quantity of water to apply. Effects of air and leaf temperature, relative humidity, illumination and wind on turgor pressure can be monitored very sensitively both under indoor and outdoor conditions. Even the effects of blue and red light as well as of oscillations of stomata aperture on turgor pressure can be monitored by the probe with high sensitivity. Similarly, water deficit due to increase of the osmotic pressure in the nutrition solutions resulted in significant changes of the probe signals. Multiple probe readings open up new possibilities to resolve (together with other techniques) the mechanisms of short- and long-distance water transport, particularly how plants can cope with water shortage. The applications of the magnetic probe are numerous and one can expect highly interesting developments in plant water relations in the nearest future.
Kuger, S., Graus, D., Brendtke, R., Gunther, N., Katzer, A., Lutyj, P., Polat, B., Chatterjee, M., Sukhorukov, V.L., Flentje, M., Djuzenova, C.S.: Radiosensitization of Glioblastoma Cell Lines by the Dual PI3K and mTOR Inhibitor NVP-BEZ235 Depends on Drug-Irradiation Schedule.Transl Oncol.6,169-179 (2013).
Previous studies have shown that the dual phosphatidylinositide 3-kinase/mammalian target of rapamycin (PI3K/mTOR) inhibitor NVP-BEZ235 radiosensitizes tumor cells if added shortly before ionizing radiation (IR) and kept in culture medium thereafter. The present study explores the impact of inhibitor and IR schedule on the radiosensitizing ability of NVP-BEZ235 in four human glioblastoma cell lines. Two different drug-IR treatment schedules were compared. In schedule I, cells were treated with NVP-BEZ235 for 24 hours before IR and the drug was removed before IR. In schedule II, the cells were exposed to NVP-BEZ235 1 hour before, during, and up to 48 hours after IR. The cellular response was analyzed by colony counts, expression of marker proteins of the PI3K/AKT/mTOR pathway, cell cycle, and DNA damage. We found that under schedule I, NVP-BEZ235 did not radiosensitize cells, which were mostly arrested in G1 phase during IR exposure. In addition, the drug-pretreated and irradiated cells exhibited less DNA damage but increased expressions of phospho-AKT and phospho-mTOR, compared to controls. In contrast, NVP-BEZ235 strongly enhanced the radiosensitivity of cells treated according to schedule II. Possible reasons of radiosensitization by NVP-BEZ235 under schedule II might be the protracted DNA repair, prolonged G2/M arrest, and, to some extent, apoptosis. In addition, the PI3K pathway was downregulated by the NVP-BEZ235 at the time of irradiation under schedule II, as contrasted with its activation in schedule I. We found that, depending on the drug-IR schedule, the NVP-BEZ235 can act either as a strong radiosensitizer or as a cytostatic agent in glioblastoma cells.
Hartmann, S., Günther, N., Biehl, M., Katzer, A., Kuger, S., Worschech, E., Sukhorukov, V.L., Krohne, G., Zimmermann, H., Flentje, M., Djuzenova, C.S.: Hsp90 inhibition by NVP-AUY922 and NVP-BEP800 decreases migration and invasion of irradiated normoxic and hypoxic tumor cell lines.Cancer Letters.331,200 - 210 (2013).
This study explores the impact of Hsp90 inhibitors NVP-AUY922 and NVP-BEP800 in combination with ionizing radiation (IR) on the migration and invasion of lung carcinoma A549 and glioblastoma SNB19 cells, under normoxia or hypoxia. Independent of oxygen concentration, both drugs decreased the migration and invasion rates of non-irradiated tumor cells. Combined drug-IR treatment under hypoxia inhibited cell invasion to a greater extent than did each treatment alone. Decreased migration of cells correlated with altered expression of several matrix-associated proteins (FAK/p-FAK, Erk2, RhoA) and impaired F-actin modulation. The anti-metastatic efficacy of the Hsp90 inhibitors could be useful in combinational therapies of cancer.
Andronic, J., Bobak, N., Bittner, S., Ehling, P., Kleinschnitz, C., Herrmann, A.M., Zimmermann, H., Sauer, M., Wiendl, H., Budde, T., Meuth, S.G., Sukhorukov, V.L.: Identification of two-pore domain potassium channels as potent modulators of osmotic volume regulation in human T lymphocytes.Biochimica et Biophysica Acta (BBA) - Biomembranes.1828,699 - 707 (2013).
Many functions of T lymphocytes are closely related to cell volume homeostasis and regulation, which utilize a complex network of membrane channels for anions and cations. Among the various potassium channels, the voltage-gated KV1.3 is well known to contribute greatly to the osmoregulation and particularly to the potassium release during the regulatory volume decrease (RVD) of T cells faced with hypotonic environment. Here we address a putative role of the newly identified two-pore domain (K2P) channels in the RVD of human CD4+ T lymphocytes, using a series of potent well known channel blockers. In the present study, the pharmacological profiles of RVD inhibition revealed K2P5.1 and K2P18.1 as the most important K2P channels involved in the RVD of both naïve and stimulated T cells. The impact of chemical inhibition of K2P5.1 and K2P18.1 on the RVD was comparable to that of KV1.3. K2P9.1 also notably contributed to the RVD of T cells but the extent of this contribution and its dependence on the activation status could not be unambiguously resolved. In summary, our data provide first evidence that the RVD-related potassium efflux from human T lymphocytes relies on K2P channels.
Ehrenberger, W., Rüger, S., Rodríguez-Domínguez, C.M., Díaz-Espejo, A., Fernández, J.E., Moreno, J., Zimmermann, D., Sukhorukov, V.L., Zimmermann, U.: Leaf patch clamp pressure probe measurements on olive leaves in a nearly turgorless state.Plant Biology.14,666--674 (2012).
The non-invasive leaf patch clamp pressure (LPCP) probe measures the attenuated pressure of a leaf patch, Pp, in response to an externally applied magnetic force. Pp is inversely coupled with leaf turgor pressure, Pc, i.e. at high Pc values the Pp values are small and at low Pc values the Pp values are high. This relationship between Pc and Pp could also be verified for 2-m tall olive trees under laboratory conditions using the cell turgor pressure probe. When the laboratory plants were subjected to severe water stress (Pc dropped below ca. 50 kPa), Pp curves show reverse diurnal changes, i.e. during the light regime (high transpiration) a minimum Pp value, and during darkness a peak Pp value is recorded. This reversal of the Pp curves was completely reversible. Upon watering, the original diurnal Pp changes were re-established within 2–3 days. Olive trees in the field showed a similar turnover of the shape of the Pp curves upon drought, despite pronounced fluctuations in microclimate. The reversal of the Pp curves is most likely due to accumulation of air in the leaves. This assumption was supported with cross-sections through leaves subjected to prolonged drought. In contrast to well-watered leaves, microscopic inspection of leaves exhibiting inverse diurnal Pp curves revealed large air-filled areas in parenchyma tissue. Significantly larger amounts of air could also be extracted from water-stressed leaves than from well-watered leaves using the cell turgor pressure probe. Furthermore, theoretical analysis of the experimental Pp curves shows that the propagation of pressure through the nearly turgorless leaf must be exclusively dictated by air. Equations are derived that provide valuable information about the water status of olive leaves close to zero Pc.
CS, D., C, B., K, R., S, K., A, K., N, N., N, G., B, P., VL, S., M, F.: Hsp90 inhibitor NVP-AUY922 enhances radiation sensitivity of tumor cell lines under hypoxia.Cancer Biol Ther.13,425-434 (2012).
NVP-AUY922, a novel inhibitor of Hsp90, was shown to enhance the effect of ionizing radiation (IR) on tumor cells under normoxic conditions. Since low oxygen tension is a common feature of solid tumors, we explore in the present study the impact of hypoxia on the combined treatment of lung carcinoma A549 and glioblastoma SNB19 cell lines with NVP-AUY922 and IR. Cellular analysis included the colony-forming ability, expression of CAIX, Hsp90, Hsp70, Raf-1, Akt, cell cycle progression and associated proteins, as well as DNA damage measured by histone gammaH2AX. The clonogenic assay revealed that in both cell lines NVP-AUY922 enhanced the radiotoxicity under hypoxic exposure to a level similar to that observed under oxic conditions. Irrespective of oxygen supply during drug treatment, NVP-AUY922 also reduced the expression of anti-apoptotic proteins Raf-1 and Akt. As judged by the levels of histone gammaH2AX, drug-treated hypoxic cells exhibited a lower repair rate of DNA double-strand breaks than normoxic cells. The drug-IR mediated changes in the cell cycle, i.e., S-phase depletion and G 2/M arrest, developed not directly during hypoxic exposure but first upon 24 h reoxygenation. Under both oxygen tensions, Hsp90 inhibition downregulated the cell cycle-associated proteins, Cdk1, Cdk4 and pRb. The finding that NVP-AUY922 can enhance the in vitro radiosensitivity of hypoxic tumor cells may have implications for the combined modality treatment of solid tumors.
Niewidok, N., Wack, L.-J., Schiessl, S., Stingl, L., Katzer, A., Polat, B., Sukhorukov, V.L., Flentje, M., Djuzenova, C.S.: Hsp90 inhibitors, NVP-AUY922 and NVP-BEP800 may exert a significant radiosensitization on tumor cells along with a cell type-specific cytotoxicity.Transl. Oncology.5,356-369 (2012).
Targeting Hsp90 provides a promising therapeutic approach to enhance the sensitivity of tumor cells to ionizing radiation (IR). To explore the impact of scheduling drug-IR administration, in the present study the response of lung carcinoma A549 and glioblastoma SNB19 cells to simultaneous drug-IR treatment followed by a long-term drug administration is analyzed. Cellular response was evaluated at different time intervals (0.5, 24 and 48 h) after IR-alone, drug-alone, or combined drug-IR treatments by colony counts, expression profiles of Hsp90 and its clients, along with several apoptotic markers and cell cycle-related proteins, as well as by IR/drug-induced cell cycle arrest, DNA damage and repair. A short 30-min exposure to either Hsp90 inhibitor did not affect the radiosensitivity of both tumor cell lines. Increasing the duration of post-IR drug treatment progressively enhanced the sensitivity of SNB19 cells to IR. In contrast, the response of A549 cells to drug-IR combination was largely determined by the cytotoxic effects of both drugs without radiosensitization. Combined drug-IR treatment induced more severe DNA damage in both tumor cell lines than each treatment alone, and also protracted the kinetics of DNA damage repair in SNB19 cells. In addition to large cell cycle disturbances, drug-IR treatment also caused depletion of the anti-apoptotic proteins Akt and Raf-1 in both cell lines, along with a decrease of survivin in A549 cells. The data show that, simultaneous Hsp90 inhibition and irradiation may induce cell-type specific radiosensitization as well as cytotoxicity against tumor cells.
Terpitz, U., Letschert, S., Bonda, U., Spahn, C., Guan, C., Sauer, M., Zimmermann, U., Bamberg, E., Zimmermann, D., Sukhorukov, V.: Dielectric Analysis and Multi-cell Electrofusion of the Yeast Pichia pastoris for Electrophysiological Studies.Journal of Membrane Biology.1-12 (2012).
The yeast Pichia pastoris has become the most favored eukaryotic host for heterologous protein expression. P. pastoris strains capable of overexpressing various membrane proteins are now available. Due to their small size and the fungal cell wall, however, P. pastoris cells are hardly suitable for direct electrophysiological studies. To overcome these limitations, the present study aimed to produce giant protoplasts of P. pastoris by means of multi-cell electrofusion. Using a P. pastoris strain expressing channelrhodopsin-2 (ChR2), we first developed an improved enzymatic method for cell wall digestion and preparation of wall-less protoplasts. We thoroughly analyzed the dielectric properties of protoplasts by means of electrorotation and dielectrophoresis. Based on the dielectric data of tiny parental protoplasts (2–4 μm diameter), we elaborated efficient electrofusion conditions yielding consistently stable multinucleated protoplasts of P. pastoris with diameters of up to 35 μm. The giant protoplasts were suitable for electrophysiological measurements, as proved by whole-cell patch clamp recordings of light-induced, ChR2-mediated currents, which was impossible with parental protoplasts. The approach presented here offers a potentially valuable technique for the functional analysis of low-signal channels and transporters, expressed heterologously in P. pastoris and related host systems.
SHIRAKASHI, R., SUKHORUKOV, V.L., REUSS, R., SCHULZ, A., ZIMMERMANN, U.: Effects of a Pulse Electric Field on Electrofusion of Giant Unilamellar Vesicle (GUV)-Jurkat Cell.Journal of Thermal Science and Technology.7,589-602 (2012).
Here we describe a new method to deliver membrane impermeable cryo-/lyo-protective agents (CPAs) into the cytosol of living cells via their electrofusion with Giant Unilamellar Vesicles (GUVs) containing large amounts of CPAs. Membrane electrofusion is commonly believed to be triggered by the irreversible electrical breakdown of the membrane at contact region induced by a DC field pulse. Therefore, analysis of the temporal changes of the membrane potential distribution in a cell-GUV pair is is necessary to achieve optimum electrofusion conditions with respect to the field pulse strength and duration. In this study, the GUV-Jurkat cell electrofusion rates under various pulse strength and length were measured. In addition, we calculated the transient membrane potentials in a deformed GUV-Jurkat pair during the electric field application by a finite element method (FEM)-electric field analysis. The relevant electric properties of both fusion partners reported previously were used for the analysis to validate the quantitative calculation results. Both experimental results and theoretical calculation suggest that; 1) GUV-Jurkat cell pairs should be stretched by AC field prior to electrofusion, 2) the whole membrane contact zone should undergo electrical breakdown at the same time to accomplish electrofusion, 3) after applying an electric field for around 1µsec, membrane potential in contact region becomes homogeneous, 4) after applying an electric field for around 10µsec, membrane potential is saturated, 5) the irreversible breakdown occurs at a membrane voltage of about 3V.
Shirakashi, R., Mischke, M., Fischer, P., Memmel, S., Krohne, G., Fuhr, G.R., Zimmermann, H., Sukhorukov, V.L.: Changes in the dielectric properties of medaka fish embryos during development, studied by electrorotation.Biochemical and biophysical research communications. (2012).
The Japanese medaka fish, Oryzias latipes, has become a powerful vertebrate model organism in developmental biology and genetics. The present study explores the dielectric properties of medaka embryos during prehatching development by means of the electrorotation (ROT) technique. Due to their layered structure, medaka eggs exhibited up to three ROT peaks in the kHz-MHz frequency range. During development from blastula to early somite stage, ROT spectra varied only slightly. But as the embryo progressed to the late-somite stage, the ROT peaks underwent significant changes in frequency and amplitude. Using morphological data obtained by light and electron microscopy, we analyzed the ROT spectra with a three-shell dielectric model that accounted for the major embryonic compartments. The analysis yielded a very high value for the ionic conductivity of the egg shell (chorion), which was confirmed by independent osmotic experiments. A relatively low capacitance of the yolk envelope was consistent with its double-membrane structure revealed by transmission electron microscopy. Yolk-free dead eggs exhibited only one co-field ROT peak, shifted markedly to lower frequencies with respect to the corresponding peak of live embryos. The dielectric data may be useful for monitoring the development and changes in fish embryos' viability/conditions in basic research and industrial aquaculture.
Rodriguez-Dominguez, C.M., Ehrenberger, W., Sann, C., Rüger, S., Sukhorukov, V., Martín-Palomo, M.J., Diaz-Espejo, A., Cuevas, M.V., Torres-Ruiz, J.M., Perez-Martin, A., Zimmermann, U., Fernández, J.E.: Concomitant measurements of stem sap flow and leaf turgor pressure in olive trees using the leaf patch clamp pressure probe.Agricultural Water Management.114,50 - 58 (2012).
Stem sap flow (Q) and leaf turgor pressure (Pc) were measured simultaneously on 4-year-old, 2.4 m tall ‘Arbequina’ olive trees in a hedgerow orchard. Measurements were performed on well-watered control trees as well as on 60RDI and 30RDI trees (RDI = regulated deficit irrigation). The 60RDI trees received 59.2% of the crop water needs (ETc), and the 30RDI trees received 29.4% of ETc. Pc was determined non-invasively using the magnetic leaf patch clamp pressure probe (ZIM probe). The patch pressure Pp measured by the probe is inversely correlated with turgor pressure at Pc > ca. 50 kPa. Pc is coupled with xylem pressure; thus Pp yields information about the development of tension in xylem. In the case of the control trees a positive correlation between Q and Pp was generally found, i.e. Q increased usually with increasing Pp and decreased with decreasing Pp, as expected. However, Q peaking did not always coincided with Pp peaking at noon. Occasionally, Q peaking preceded or followed Pp peaking with a time difference of up to 3 h in both cases. Under some circumstances, the onset of Q after sunrise was greatly delayed, even though a pronounced increase of Pp was observed. A delayed onset of Q after sunrise resulted in hysteresis phenomena, i.e. the linear increase of Q and Pp in the morning hours did not coincide with the corresponding decrease of Q and Pp in the afternoon. The development of severe water stress (Pc < ca. 50 kPa) associated with the increase in the intercellular spaces of the spongy tissue in the leaves resulted in inverted diurnal Pp curves, i.e. minimum Pp values were recorded at noon and maximum Pp values during the night on the 30RDI trees. The effects were reversible as shown by re-watering. By contrast, the magnitude of Q decreased continuously from the turgescent state to the state of severe water stress; maximum Q values were still recorded around noon. The data suggests that short-range tension forces are responsible for water lifting in olive trees and that water uptake from water storage reservoirs must play an important role in the supply of the leaves with water. Furthermore, for setting of irrigation thresholds the finding of shape changes of the Pp curves upon severe water stress seems to be a useful indicator. Such shape changes are detected and monitored more sensitively than changes in the magnitude of sap flow rates or of turgor pressure.
Niewidok, N., Wack, L.-J., Stingl, L., Katzer, A., Sukhorukov, V.L., Flentje, M., Djuzenova, C.S.: Simultaneous radiation and Hsp90 inhibition by NVP-AUY922 or NVP-BEP800 is more efficient in tumor cell killing than drug-first modality followed by irradiation.STRAHLENTHERAPIE UND ONKOLOGIE.187,125-126 (2011).
SHIRAKASHI, R., REUSS, R., SCHULZ, A., SUKHORUKOV, V.L., ZIMMERMANN, U.: Effects of a Pulse Electric Field on Electrofusion of Giant Unilamellar Vesicle (GUV) -Jurkat Cell (Measurement of Fusion Ratio and Electric Field Analysis of Pulsed GUV-Jurkat Cell).TRANSACTIONS OF THE JAPAN SOCIETY OF MECHANICAL ENGINEERS Series B.77,1269-1278 (2011).
Electrofusion of Giant Unilamellar Vesicle (GUV) and living cell is a gentle method to deliver membrane impermeable cryo-/lyo-protective molecules into living cell for cryo-/lyopreservation. In this method, cellular fusion is believed to be triggered by the irreversible electical breakdown of the membrane at contact region. Nevertheless, as the time change of the membrane potential distribution during an electric pulse is still not clear, the length and strength of electric pulse are hardly designed. In this study, the GUV-Jurkat electrofusion ratios under various pulse strength and length were measured. In addition, we calculated the time change of membrane potential distribution of a deformed GUV-Jurkat pair during the electric field application by a finite element method (FEM)-electric field analysis. The measured cellular and GUV's electric properties and a GUV-Jurkat profile were used for the analysis to validate the quantitative calculation results. Both results of experiment and calculation suggested that; 1) GUV-Jurkat should be stretched for the electrofusion, 2) whole contact region of membrane should breakdown at the same time to process electrofusion, 3) after applying an electric field for around 1μsec, membrane potential in contact region becomes homogeneous, 4) after applying an electric field for around 10μsec, membrane potential is relaxed, 5) the irreversible electric breakdown is around 3V.
Bobak, N., Bittner, S., Andronic, J., Hartmann, S., Mühlpfordt, F., Schneider-Hohendorf, T., Wolf, K., Schmelter, C., Göbel, K., Meuth, P., Zimmermann, H., Döring, F., Wischmeyer, E., Budde, T., Wiendl, H., Meuth, S.G., Sukhorukov, V.L.: Volume regulation of murine T lymphocytes relies on voltage-dependent and two-pore domain potassium channels.Biochimica et Biophysica Acta (BBA) - Biomembranes.1808,2036 - 2044 (2011).
A variety of ion channels are supposed to orchestrate the homoeostatic volume regulation in T lymphocytes. However, the relative contribution of different potassium channels to the osmotic volume regulation and in particular to the regulatory volume decrease (RVD) in T cells is far from clear. This study explores a putative role of the newly identified K2P channels (TASK1, TASK2, TASK3 and TRESK) along with the voltage-gated potassium channel KV1.3 and the calcium-activated potassium channel KCa3.1 in the RVD of murine T lymphocytes, using genetic and pharmacological approaches. K2P channel knockouts exerted profound effects on the osmotic properties of murine T lymphocytes, as revealed by reduced water and RVD-related solute permeabilities. Moreover, both genetic and pharmacological data proved a key role of KV1.3 and TASK2 channels in the RVD of murine T cells exposed to hypotonic saline. Our experiments demonstrate a leading role of potassium channels in the osmoregulation of T lymphocytes under different conditions. In summary, the present study sheds new light on the complex and partially redundant network of potassium channels involved in the basic physiological process of the cellular volume homeostasis and extends the repertoire of potassium channels by the family of K2P channels.
Storz, H., Mueller, K.J., Ehrhart, F., Gomez, I., Shirley, S.G., Gessner, P., Zimmermann, G., Weyand, E., Sukhorukov, V.L., Forst, T., Weber, M.M., Zimmermann, H., Kulicke, W.-M., Zimmermann, U.: Physicochemical features of ultra-high viscosity alginates.CARBOHYDRATE RESEARCH.344,985-995 (2009).
The physicochemical characteristics of the ultra-high viscosity and highly biocompatible alginates extracted from Lessonia nigrescens (UHVN) and Lessonia trabeculata (UHVT) were analyzed. Fluorescence and H-1 NMR spectroscopies, viscometry, and multi-angle light scattering (MALS) were used for elucidation of the chemical structure, molar mass, and coil size. The sequential structures from NMR spectroscopy showed high guluronate content for UHVT, but low for UHVN. Intrinsic viscosity [eta] measurements exhibited unusual high values (up to 2750 mL/g), whereas [eta] of a commercial alginate was only about 970 mL/g. MALS batch measurements of the UHV-alginates yielded ultra-high values of the weight average molar mass (M-w up to 1.1 x 10(6) g/mol) and of the z-average gyration radius (< R-G >(z) up to 191 nm). The M-w and < R-G >(z) distributions of UHV-alginates and of ultrasonically degraded fractions were determined using size exclusion chromatography combined with MALS and asymmetrical flow-field-flow fractionation. The M-w dependency of [eta] and < RG >(z) could be described by [eta] = 0.059 x M-w(0.78) and < R-G >(z) = 0.103 x M-w(x). (UHVN: x= 0.52: UHVT: x = 0.53) indicating that the monomer composition has no effect on coil expansion. Therefore, the equations can be used to calculate M-w and < R-G >(z) Values of UHVT- and UHVN-alginate mixtures as used in immunoisolation. Furthermore, the simple and inexpensive capillary viscometry can be used for real-time validation of the extraction and purification process of the UHV-alginates. (C) 2009 Elsevier Ltd. All rights reserved.
Djuzenova, C.S., Krasnyanska, J., Kiesel, M., Stingl, L., Zimmermann, U., Flentje, M., Sukhorukov, V.L.: Intracellular delivery of 2-deoxy-D-glucose into tumor cells by long-term cultivation and through swelling-activated pathways: Implications for radiation treatment.MOLECULAR MEDICINE REPORTS.2,633-640 (2009).
2-Deoxy-D-glucose (2DG), a well-known inhibitor of anaerobic glycolysis, is expected to exert cytotoxic and radiosensitizing effects. In order to test this hypothesis, the response of four tumor cell lines (U87-MG, GaMG, A549 and HT1080) to 2DG was analyzed for cell proliferation, changes in cell volume and nucleus size, as well as for radiation-induced DNA fragmentation, measured by the alkaline Comet assay. Two methods were used for loading cells with 2DG. The Ion-term method included cell cultivation in the presence of 5 mM 2DG for 24 h, while rapid intracellular delivery of 2DG was achieved by exposing the cells for 20 min to a hypotonic solution containing 100 mM 2DG. Irrespective of the loading method, 2DG inhibited the growth of HT1080 and A549 cells. In contrast, two glioblastoma lines (U87 and GaMG) were resistant to 2DG. In three of the four cell lines (all except HT1080), long-term treatment with 2DG reduced radiation-induced DNA fragmentation in conjunction with 2DG-mediated nucleus shrinkage (probably via chromatin condensation) in non-irradiated cells. Complementary volumetric experiments revealed the avid hypotonic uptake of 2DG by all tumor lines. Nonetheless, only HT1080 cells exhibited a significant increase in radiation-induced DNA fragmentation upon hypotonic loading with 2DG, associated with marked nucleus expansion in non-irradiated samples. Our data suggest that, dependant on cell type as well as on medium composition and tonicity, sugar treatment can induce the compaction or expansion of chromatin, thus decreasing or increasing radiation-induced DNA fragmentation. These results raise interesting questions for further studies on the mechanistic links between the sugar-modulated cell volume changes, chromatin structure and radiosensitivity of tumor and normal cells.
Sukhorukov, V.L., Imes, D., Woellhaf, M.W., Andronic, J., Kiesel, M., Shirakashi, R., Zimmermann, U., Zimmermann, H.: Pore size of swelling-activated channels for organic osmolytes in Jurkat lymphocytes, probed by differential polymer exclusion.BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES.1788,1841-1850 (2009).
The present study explores the impact of the molecular size on the permeation of low-molecular-weight polyethylene glycols (PEG200-1500) through the plasma membrane of Jurkat cells under iso- and hypotonic conditions. To this end, we analyzed the cell volume responses to PEG-substituted solutions of different osmolalities (100-300 mOsm) using video microscopy. In parallel experiments, the osmotically induced changes in the membrane capacitance and cytosolic conductivity were measured by electrorotation (ROT). Upon moderate swelling in slightly hypotonic solutions (200 mOsm), the lymphocyte membrane remained impermeable to PEG300-1500, which allowed the cells to accomplish regulatory volume decrease (RVD). During RVD, lymphocytes released intracellular electrolytes through the swelling-activated pathways, as proved by a decrease of the cytosolic conductivity measured by electrorotation. RVD also occurred in strongly hypotonic solutions (100 mOsm) of PEG600-1500, whereas 100 mOsm solutions of PEG300-400 inhibited RVD in Jurkat cells. These findings suggest that extensive hypotonic swelling rendered the cell membrane highly permeable to PEG300-400, but not to PEG600-1500. The swelling-activated channels conducting PEG300-400 were inserted into the plasma membrane from cytosolic vesicles via swelling-mediated exocytosis, as suggested by an increase of the whole cell capacitance. Using the hydrodynamic radii R-h Of PEGs (determined by viscosimetry), the observed size-selectivity of membrane permeation yielded an estimate of similar to 0.74 nm for the cut-off radius of the swelling-activated channel for organic osmolytes. Unlike PEG300-1500, the smallest PEG (PEG200, R-h = 0.5 nm) permeated the lymphocyte membrane under isotonic conditions thus leading to a continuous isotonic swelling. The results are of interest for biotechnology and biomedicine, where PEGs are widely used for cryopreservation of cells and tissues. (C) 2009 Elsevier B.V. All rights reserved.
Terpitz, U., Raimunda, D., Westhoff, M., Sukhorukov, V.L., Beauge, L., Bamberg, E., Zimmermann, D.: Electrofused giant protoplasts of Saccharomyces cerevisiae as a novel system for electrophysiological studies on membrane proteins.BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES.1778,1493-1500 (2008).
Giant protoplasts of Saccharomyces cerevisiae of 10-35 mu m in diameter were generated by multi-cell electrofusion. Thereby two different preparation strategies were evaluated with a focus on size distribution and ``patchability'' of electrofused protoplasts. In general, parental protoplasts were suitable for electrofusion 1-12 h after isolation. The electrophysiological properties of electrofused giant protoplasts could be analyzed by the whole-cell patch clamp technique. The area-specific membrane capacitance (0.66 +/- 0.07 mu F/cm(2)) and conductance (23-44 mu S/cm(2)) of giant protoplasts were consistent with the corresponding data for parental protoplasts. Measurements with fluorescein-filled patch pipettes allowed to exclude any internal compartmentalisation of giant protoplasts by plasma membranes, since uniform (diffusion-controlled) dye uptake was only observed in the whole-cell configuration, but not in the cell-attached formation. The homogeneous structure of giant protoplasts was further confirmed by the observation that no plasma membrane associated fluorescence was seen in the interior of giant cells after electrofusion of protoplasts expressing the light-activated cation channel Channelrhodopsin-2 (ChR2) linked to yellow fluorescent protein (YFP). Patch clamp analysis of the heterologously expressed ChR2-YFP showed typical blue light dependent, inwardly-directed currents for both electrofused giant and parental protoplasts. Most importantly, neither channel characteristics nor channel expression density was altered by electric field treatment. Summarising, multi-cell electrofusion increases considerably the absolute number of membrane proteins accessible in patch clamp experiments, thus presumably providing a convenient tool for the biophysical investigation of low-signal transporters and channels. (C) 2008 Elsevier B.V. All rights reserved.
Zimmermann, D., Zhou, A., Kiesel, M., Feldbauer, K., Terpitz, U., Haase, W., Schneider-Hohendorf, T., Bamberg, E., Sukhorukov, V.L.: Effects on capacitance by overexpression of membrane proteins.BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS.369,1022-1026 (2008).
Functional Channelrhodopsin-2 (ChR2) overexpression of about 10(4) channels/mu m(2) in the plasma membrane of HEK293 cells was studied by patch-clamp and freeze-fracture electron microscopy. Simultaneous electrorotation measurements revealed that ChR2 expression was accompanied by a marked increase of the area-specific membrane capacitance (C-m). The C-m increase apparently resulted partly from an enlargement of the size and/or number of microvilli. This is suggested by a relatively large C-m of 1.15 +/- 0.08 mu F/cm(2) in ChR2-expressing; cells measured under isotonic conditions. This value was much higher than that of the control HEK293 cells (039 +/- 0.02 mu F/cm(2)). However, even after complete loss of microvilli under strong hypoosmolar conditions (100 mOsm), the ChR2-expressing cells still exhibited a significantly larger C-m (0.85 +/- 0.07 mu F/cm(2)). as compared to non-expressing control cells (0.70 +/- 0.03 mu F/cm(2)). Therefore, a second mechanism of capacitance increase may involve changes in the membrane permittivity and/or thickness due to the embedded ChR2 proteins. (C) 2008 Elsevier Inc. All rights reserved.
Zimmermann, D., Kiesel, M., Terpitz, U., Zhou, A., Reuss, R., Kraus, J., Schenk, W.A., Bamberg, E., Sukhorukov, V.L.: A combined patch-clamp and electrorotation study of the voltage- and frequency-dependent membrane capacitance caused by structurally dissimilar lipophilic anions.JOURNAL OF MEMBRANE BIOLOGY.221,107-121 (2008).
Interactions of structurally dissimilar anionic compounds with the plasma membrane of HEK293 cells were analyzed by patch clamp and electrorotation. The combined approach provides complementary information on the lipophilicity, preferential affinity of the anions to the inner/outer membrane leaflet, adsorption depth and transmembrane mobility. The anionic species studied here included the well-known lipophilic anions dipicrylamine (DPA(-)), tetraphenylborate (TPB-) and [W-2(CO)(10)(S2CH)](-), the putative lipophilic anion B(CF3)(4)(-) and three new heterocyclic W(CO)(5) derivatives. All tested anions partitioned strongly into the cell membrane, as indicated by the capacitance increase in patch-clamped cells. The capacitance increment exhibited a bell-shaped dependence on membrane voltage. The midpoint potentials of the maximum capacitance increment were negative, indicating the exclusion of lipophilic anions from the outer membrane leaflet. The adsorption depth of the large organic anions DPA(-), TPB- and B(CF3)(4)(-) increased and that of W(CO)(5) derivatives decreased with increasing concentration of mobile charges. In agreement with the patch-clamp data, electrorotation of cells treated with DPA(-) and W(CO)(5) derivatives revealed a large dispersion of membrane capacitance in the kilohertz to megahertz range due to the translocation of mobile charges. In contrast, in the presence of TPB- and B(CF3)(4)(-) no mobile charges could be detected by electrorotation, despite their strong membrane adsorption. Our data suggest that the presence of oxygen atoms in the outer molecular shell is an important factor for the fast translocation ability of lipophilic anions.
Dessie, S.-W., Rings, F., Holker, M., Gilles, M., Jennen, D., Tholen, E., Havlicek, V., Besenfelder, U., Sukhorukov, V.L., Zimmermann, U., Endter, J.M., Sirard, M.-A., Schellander, K., Tesfaye, D.: Dielectrophoretic behavior of in vitro-derived bovine metaphase II oocytes and zygotes and its relation to in vitro embryonic developmental competence and mRNA expression pattern.REPRODUCTION.133,931-946 (2007).
Selecting developmentally competent oocytes and zygotes based on their morphology is more often influenced by personal judgments and lacks universal standards. Therefore, this experiment was conducted to investigate the rate of development and mRNA level of dielectrophoretically separated oocytes and zygotes to validate dielectrophoresis (DEP) as non-invasive option for selection of oocytes and zygotes. In the first experiment, metaphase 11 oocytes with (PB+) and without (PB-) first polar body and zygotes were subjected to DEP at 4 MHz and 450 mu m electrode distance and classified into fast, very fast, slow, and very slow depending on the time elapsed to reach one of the electrodes in the electric field. Parthenogenetic activation was employed to monitor the embryonic development of dielectrophoretically classified oocytes. The result revealed that at 6 and 7 days of postactivation, the blastocyst rate of very slow dielectrophoretic PB+ and PB- oocytes was significantly (P< 0.05) lower than other groups. Similarly, in zygotes, the blastocyst rate at 7 days post-insemination was higher (P<0.05) in the very fast dielectrophoretic categories when compared with the slow and very slow categories. In the second experiment, mRNA level was analyzed in the very fast and very slow dielectrophoretic PB+ oocytes and zygotes respectively using the bovine cDNA microarray. The result showed that 36 and 42 transcripts were differentially regulated between the very fast and very slow dielectrophoretic categories PB+ oocytes and zygotes respectively. In conclusion, clielectrophoretically separated oocytes and zygotes showed difference in the rate of blastocyst development accompanied by difference in transcriptional abundances.
Sukhorukov, V.L., Endter, J.M., Zimmermann, D., Shirakashi, R., Fehrmann, S., Kiesel, M., Reuss, R., Becker, D., Hedrich, R., Bamberg, E., Roitsch, T., Zimmermann, U.: Mechanisms of electrically mediated cytosolic Ca2+ transients in Aequorin-Transformed tobacco cells.BIOPHYSICAL JOURNAL.93,3324-3337 (2007).
Cytosolic Ca2+ changes induced by electric. field pulses of 50-mu s duration and 200-800 V/cm strength were monitored by measuring chemiluminescence in aequorin-transformed BY-2 tobacco cells. In Ca2+-substituted media, electropulsing led to a very fast initial increase of the cytosolic Ca2+ concentration reaching a peak value within < 100-200 ms. Peaking of [Ca2+](cyt) was followed by a biphasic decay due to removal of Ca2+ (e. g., by binding and/or sequestration in the cytosol). The decay had fast and slow components, characterized by time constants of; similar to 0.5 and 3-5 s, respectively. Experiments with various external Ca2+ concentrations and conductivities showed that the fast decay arises from Ca2+ fluxes through the plasmalemma, whereas the slow decay must be assigned to Ca2+ fluxes through the tonoplast. The amplitude of the [Ca2+](cyt) transients increased with increasing. field strength, whereas the time constants of the decay kinetics remained invariant. Breakdown of the plasmalemma was achieved at a critical. field strength of similar to 450 V/cm, whereas breakdown of the tonoplast required similar to 580 V/cm. The above. finndings could be explained by the transient potential profiles generated across the two membranes in response to an exponentially decaying. field pulse. The dielectric data required for calculation of the tonoplast and plasmalemma potentials were derived from electrorotation experiments on isolated vacuolated and evacuolated BY-2 protoplasts. The electrorotation response of vacuolated protoplasts could be described in terms of a three-shell model (i.e., by assuming that the capacitances of tonoplast and plasmalemma are arranged in series). Among other things, the theoretical analysis together with the experimental data show that genetic manipulations of plant cells by electrotransfection or electrofusion must be performed in low-conductivity media to minimize release of vacuolar Ca2+ and presumably other vacuolar ingredients.
Zimmermann, H., Waehlisch, F., Baier, C., Westhoff, M., Reuss, R., Zimmermann, D., Behringer, M., Ehrhart, F., Katsen-Globa, A., Giese, C., Marx, U., Sukhorukov, V.L., Vasquez, J.A., Jakob, P., Shirley, S.G., Zimmermann, U.: Physical and biological properties of barium cross-linked alginate membranes.BIOMATERIALS.28,1327-1345 (2007).
We describe the manufacture of highly stable and elastic alginate membranes with good cell adhesivity and adjustable permeability. Clinical grade, ultra-high viscosity alginate is gelled by diffusion of Ba2+ followed by use of the ``crystal gun'' [Zimmermann H. et al., Fabrication of homogeneously cross-linked, functional alginate microcapsules validated by NMR-, CLSM- and AFM-imaging. Biomaterials 2003;24:2083-96]. Burst pressure of well-hydrated membranes is between 34 and 325 kPa depending on manufacture and storage details. Water flows induced by sorbitol and raffinose (probably diffusional) are lower than those caused by PEG 6000, which may be related to a Hagen-Poiseuille flow. Hydraulic conductivity, L-p, from PEG-induced flows ranges between 2.4 x 10(-12) and 6.5 x 10(-12) m Pa-1 s(-1). Hydraulic conductivity measured with hydrostatic pressure up to 6 kPa is 2-3 orders of magnitude higher and decreases with increasing pressure to about 3 x 10(-10) in Pa-1 s(-1) at 4 kPa. Mechanical introduction of 200 mu m-diameter pores increases hydraulic conductivity dramatically without loss of mechanical stability or flexibility. NMR imaging with Cu2+ as contrast agent shows a layered structure in membranes cross-linked for 2 h. Phase contrast and atomic force microscopy in liquid environment reveals surface protrusions and cavities correlating with steps of the production process. Murine L929 cells adhere strongly to the rough surface of crystal-bombarded membranes. NaCl-mediated membrane swelling can be prevented by partial replacement of salt with sorbitol allowing cell culture on the membranes. (c) 2006 Elsevier Ltd. All rights reserved.
Kiesel, M., Reuss, R., Endter, J., Zimmermann, D., Zimmermann, H., Shirakashi, R., Bamberg, E., Zimmermann, U., Sukhorukov, V.L.: Swelling-activated pathways in human T-lymphocytes studied by cell volumetry and electrorotation.BIOPHYSICAL JOURNAL.90,4720-4729 (2006).
Small organic solutes, including sugar derivatives, amino acids, etc., contribute significantly to the osmoregulation of mammalian cells. The present study explores the mechanisms of swelling-activated membrane permeability for electrolytes and neutral carbohydrates in Jurkat cells. Electrorotation was used to analyze the relationship between the hypotonically induced changes in the electrically accessible surface area of the plasma membrane ( probed by the capacitance) and its permeability to the monomeric sugar alcohol sorbitol, the disaccharide trehalose, and electrolyte. Time-resolved capacitance and volumetric measurements were performed in parallel using media of different osmolalities containing either sorbitol or trehalose as the major solute. Under mild hypotonic stress in 200 mOsm sorbitol or trehalose solutions, the cells accomplished regulatory volume decrease by releasing cytosolic electrolytes presumably through pathways activated by the swelling-mediated retraction of microvilli. This is suggested by a rapid decrease of the area-specific membrane capacitance C-m(mu F/cm(2)). The cell membrane was impermeable to both carbohydrates in 200 mOsm media. Whereas trehalose permeability remained also very poor in 100 mOsm medium, extreme swelling of cells in a strongly hypotonic solution (100 mOsm) led to a dramatic increase in sorbitol permeability as evidenced by regulatory volume decrease inhibition. The different osmotic thresholds for activation of electrolyte release and sorbitol influx suggest the involvement of separate swelling-activated pathways. Whereas the electrolyte efflux seemed to utilize pathways preexisting in the plasma membrane, putative sorbitol channels might be inserted into the membrane from cytosolic vesicles via swelling-mediated exocytosis, as indicated by a substantial increase in the whole-cell capacitance C-C (pF) in strongly hypotonic solutions.
Sukhorukov, V.L., Reuss, R., Endter, J.M., Fehrmann, S., Katsen-Globa, A., Gessner, P., Steinbach, A., Mueller, K.J., Karpas, A., Zimmermann, U., Zimmermann, H.: A biophysical approach to the optimisation of dendritic-tumour cell electrofusion.BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS.346,829-839 (2006).
Electrofusion of tumour and dendritic cells (DCs) is a promising approach for production of DC-based anti-tumour vaccines. Although human DCs are well characterised immunologically, little is known about their biophysical properties, including dielectric and osmotic parameters, both of which are essential for the development of efficient electrofusion protocols. In the present study, human DCs from the peripheral blood along with a tumour cell line used as a model fusion partner were examined by means of time-resolved cell volumetry and elect to rotation. Based on the biophysical cell data, the electrofusion protocol could be rapidly optimised with respect to the sugar composition of the fusion medium, duration of hypotonic treatment, frequency range for stable cell alignment, and field strengths of breakdown pulses triggering membrane fusion. The hypotonic electrofusion consistently gave a tumour-DC hybrid rate of up to 19%. as determined by counting dually labelled fluorescent hybrids in a microscope. This fusion rate is nearly twice as high as that usually reported in the literature for isotonic media. The experimental findings and biophysical approach presented here are generally useful for the development of efficient electrofusion protocols, especially for rare and valuable human cells. (c) 2006 Elsevier Inc. All rights reserved.
Zimmermann, D., Terpitz, U., Zhou, A., Reuss, R., Mueller, K., Sukhorukov, V.L., Gessner, P., Nagel, G., Zimmermann, U., Bamberg, E.: Biophysical characterisation of electrofused giant HEK293-cells as a novel electrophysiological expression system.BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS.348,673-681 (2006).
Giant HEK293 cells of 30-65 mu m in diameter were produced by three-dimensional multi-cell electrofusion in 75 mOsm sorbitol media. These strong hypotonic conditions facilitated fusion because of the spherical shape and smooth membrane surface of the swollen cells. A regulatory volume decrease (RVD), as observed at higher osmolalities, did not occur at 75 mOsm. In contrast to field-treated, but unfused cells, the increase in volume induced by hypotonic shock was only partly reversible in the case of fused giant cells after their transfer into isotonic medium. The large size of the electrofused cells allowed the study of their electrophysiological properties by application of both whole-cell and giant excised patch-clamp techniques. Recordings on giant cells yielded a value of 1.1 +/- 0.1 mu F/cm(2) for the area-specific membrane capacitance. This value was consistent with that of the parental cells. The area-specific conductivity of giant cells (diameter > 50 mu m) was found to be between 12.8 and 16.1 mu S/cm(2), which is in the range of that of the parental cells. Measurements with patch-pipettes containing fluorescein showed uniform dye uptake in the whole-cell configuration, but not in the cell-attached configuration. The diffusion-controlled uniform uptake of the dye into the cell interior excludes internal compartmentalisation. The finding of a homogeneous fusion was also supported by expression of the yellow fluorescent protein YFP (as part of the fusion-protein ChR2-YFP) in giant cells since no plasma-membrane bound YFP-mediated fluorescence was detected in the interior of the electrofused cells. Functional expression and the electrophysiological characterisation of the light-activated cation channel Channelrhodopsin 2 (ChR2) yielded similar results as for parental cells. Most importantly, the giant cells exhibited a comparable expression density of the channel protein in the plasma membrane as observed in parental cells. This demonstrates that electrofused cells can be used as a heterologous expression system. (c) 2006 Elsevier Inc. All rights reserved.
Zimmermann, H., Zimmermann, D., Reuss, R., Feilen, P.J., Manz, B., Katsen, A., Weber, M., Ihmig, F.R., Ehrhart, F., Gessner, P., Behringer, M., Steinbach, A., Wegner, L.H., Sukhorukov, V.L., Vasquez, J.A., Schneider, S., Weber, M.M., Volke, F., Wolf, R., Zimmermann, U.: Towards a medically approved technology for alginate-based microcapsules allowing long-term immunoisolated transplantation.JOURNAL OF MATERIALS SCIENCE-MATERIALS IN MEDICINE.16,491-501 (2005).
The concept of encapsulated-cell therapy is very appealing, but in practice a great deal of technology and know-how is needed for the production of long-term functional transplants. Alginate is one of the most promising biomaterials for immunoisolation of allogeneic and xenogeneic cells and tissues (such as Langerhans islets). Although great advances in alginate-based cell encapsulation have been reported, several improvements need to be made before routine clinical applications can be considered. Among these is the production of purified alginates with consistently high transplantation-grade quality. This depends to a great extent on the purity of the input algal source as well as on the development of alginate extraction and purification processes that can be validated. A key engineering challenge in designing immunoisolating alginate-based microcapsules is that of maintaining unimpeded exchange of nutrients, oxygen and therapeutic factors (released by the encapsulated cells), while simultaneously avoiding swelling and subsequent rupture of the microcapsules. This requires the development of efficient, validated and well-documented technology for cross-linking alginates with divalent cations. Clinical applications also require validated technology for long-term cryopreservation of encapsulated cells to maintaining a product inventory in order to meet end-user demands. As shown here these demands could be met by the development of novel, validated technologies for production of transplantation-grade alginate and microcapsule engineering and storage. The advances in alginate-based therapy are demonstrated by transplantation of encapsulated rat and human islet grafts that functioned properly for about 1 year in diabetic mice. (C) 2005 Springer Science + Business Media, Inc.
Sukhorukov, V.L., Reuss, R., Zimmermann, D., Held, C., Muller, K.J., Kiesel, M., Gessner, P., Steinbach, A., Schenk, W.A., Bamberg, E., Zimmermann, U.: Surviving high-intensity field pulses: Strategies for improving robustness and performance of electrotransfection and electrofusion.JOURNAL OF MEMBRANE BIOLOGY.206,187-201 (2005).
Electrotransfection and electrofusion, both widely used in research and medical applications, still have to face a range of problems, including the existence of electroporation-resistant cell types, cell mortality and also great batch-to-batch variations of the transfection and fusion yields. In the present study, a systematic analysis of the parameters critical for the efficiency and robustness of electromanipulation protocols was performed on five mammalian cell types. Factors examined included the sugar composition of hypotonic pulse media (trehalose, sorbitol or inositol), the kinetics of cell volume changes prior to electropulsing, as well as the growth medium additives used for post-pulse cell cultivation. Whereas the disaccharide trehalose generally allowed regulatory volume decrease (RVD), the monomeric sugar alcohols sorbitol and inositol inhibited RVD or even induced secondary swelling. The different volume responses could be explained by the sugar selectivity of volume-sensitive channels (VSC) in the plasma membrane of all tested cell types. Based on the volumetric data, highest transfection and fusion yields were mostly achieved when the target cells were exposed to hypotonicity for about 2 min prior to electropulsing. Longer hypotonic treatment (10-20 min) decreased the yields of viable transfected and hybrid cells due to (1) the cell size reduction upon RVD (trehalose) or (2) the excessive losses of cytosolic electrolytes through VSC (inositol/sorbitol). Doping the plasma membrane with lipophilic anions prevented both cell shrinkage and ion losses (probably due to VSC inhibition), which in turn resulted in increased transfection and fusion efficiencies.
Reuss, R., Horbaschek, M., Endter, J.M., Zimmermann, U., Sukhorukov, V.L.: The effect of disaccharides on the transport of lipophilic ions in cell membranes studied by electrorotation.ELECTROSTATICS 2003.101-106 (2004).
The disaccharide trehalose, a natural cryoprotectant, is increasingly being exploited in biomedicine as a very efficient stabilizer of frozen and dry macromolecules, membranes and whole cells. Valuable insight into the mechanisms by which trehalose protects cells can be obtained by studying its effects on the electrical and ion transport properties of cell membranes. In the present study, the binding and translocation of the lipophilic anion dipicrylamine (DPA) across the membrane of Jurkat lymphocytes (suspended in hypotonic trehalose- or sucrose-substituted media) were determined by means of electrorotation. From the rotation spectra of individual cells, not only the passive electrical properties of cell compartments but also the area-specific concentration of DPA adsorbed to the plasma membrane and its translocation rate constants were evaluated. The substitution of sucrose by trehalose increased significantly both the plasma membrane capacitance C-m and the adsorption of DPA to the plasma membrane, whereas the translocation rate of DPA across the membrane was slightly reduced. The high C-m value indicates that hypotonic stress did not cause any noticeable loss of membrane folds and microvilli in the presence of trehalose. The observed changes in the transport of DPA are consistent with the assumption that trehalose increased the dipole potential of the plasma membrane.
Shirakashi, R., Sukhorukov, V.L., Tanasawa, I., Zimmermann, U.: Measurement of the permeability and resealing time constant of the electroporated mammalian cell membranes.INTERNATIONAL JOURNAL OF HEAT AND MASS TRANSFER.47,4517-4524 (2004).
In this study a new method is presented for measuring the transient permeability of mammalian cell membranes to sugar and electrolyte molecules based on the volumetric response of cells subjected to electroporation. The time constant of membrane resealing was determined independently by flow cytometry using a fluorescent dye as the reporter molecule. The volumetric and dye uptake data were analyzed with a model relating the cell volume changes to the solute transport across the reversibly permeabilized cell membrane. The experimental approach developed here might be useful for estimating the amount of electroinjected molecules, which are difficult to measure directly. (C) 2004 Elsevier Ltd. All rights reserved.
Reuss, R., Ludwig, J., Shirakashi, R., Ehrhart, F., Zimmermann, H., Schneider, S., Weber, M.M., Zimmermann, U., Schneider, H., Sukhorukov, V.L.: Intracellular delivery of carbohydrates into mammalian cells through swelling-activated pathways.JOURNAL OF MEMBRANE BIOLOGY.200,67-81 (2004).
Volume changes of human T-lymphocytes (Jurkat line) exposed to hypotonic carbohydrate-substituted solutions of different composition and osmolality were studied by videomicroscopy. In 200 mOsm media the cells first swelled within 1-2 min and then underwent regulatory volume decrease (RVD) to their original isotonic volume within 10-15 min. RVD also occurred in strongly hypotonic 100 mOsni solutions of di- and trisaccharides (trehalose, sucrose, raffinose). In contrast to oligosaccharide media, 100 mOsm solutions of monomeric carbohydrates (glucose. galactose, mositol and sorbitol) inhibited RVD. The complex volumetric data were analyzed with a membrane transport model that allowed the estimation of the hydraulic conductivity volume-dependent Solute permeabilities and ties. We found that under slightly hypotonic stress (200 mOsm) the cell membrane was impermeable to all carbohydrates studied here. Upon osmolality decrease to 100 mOsm, the membrane permeability to monomeric carbohydrates increased dramatically (apparently due to channel activation caused by extensive cell swelling), whereas oligosaccharide permeability remained very poor. The size-selectivity of the swelling-activated sugar permeation was confirmed by direct chromatographic measurements of intracellular sugars. The results of this study are of interest for biotechnology, where sugars and related compounds are increasingly being used as potential cryo- and lyoprotective agents for preservation of rare and valuable mammalian cells and tissues.
Muller, K.J., Horbaschek, M., Lucas, K., Zimmermann, U., Sukhorukov, V.L.: Electrotransfection of anchorage-dependent mammalian cells.EXPERIMENTAL CELL RESEARCH.288,344-353 (2003).
Reversible electropermeabilization (or electroporation) of cell membranes is a very efficient method for intracellular delivery of xenomolecules, particularly of DNA. In the case of anchorage-dependent cells, however, enzymatic or mechanical detachment from the substratum is required prior to electropulsing. This can damage the plasma membrane and lead to low transfection yields. Here we present an efficient method for in situ electroporation of mammalian cells while they are attached to a solid substratum. For this purpose an electroporation chamber was constructed that housed a cell culture insert with a cell monolayer grown on a porous filter. By real-time monitoring the transmonolayer resistance, the field pulse parameters resulting in transient and reversible permeabilization of cell membranes were determined for two adherent cell lines, which were found to differ markedly in their sensitivity to electropulsing. Based on the transmonolayer resistance data, the pulsing conditions for optimum electrotransfection of two murine cell lines with plasmid DNA could be established in a very short time. The transfection yield and gene expression were significantly higher in cell monolayers facing the cathode compared to those exposed to field pulses of the reverse direction. This might be due to contribution of the electrophoresis to the translocation of the polyanionic plasmid DNA across the electropermeabilized cell membrane. The experimental setup presented here appears to be a promising tool not only for rapid optimization of in situ electrotransfection of anchorage-dependent cells but also for studying the molecular/biophysical mechanisms of the membrane breakdown and resealing. (C) 2003 Elsevier Science (USA). All rights reserved.
Schmidt, E., Kromminga, A., Kurschner, M., Zimmermann, H., Katsen, A.D., Brocker, E.B., Zillikens, D., Zimmermann, U., Sukhorukov, V.L.: Trehalose conserves expression of bullous pemphigoid antigen 180 during desiccation and freezing.JOURNAL OF IMMUNOLOGICAL METHODS.275,179-190 (2003).
Bullous pemphigoid antigen 180 (BP180) is targeted by autoantibodies in a variety of subepidermal blistering skin diseases. We have recently developed a simple, highly specific and sensitive immunofluorescence (IF) assay for the detection of circulating antibodies against BP180. This novel assay involves the expression of full-length (FL) BP180 in Sf21 insect cells that are then examined under IF microscopy after staining with anti-BP180 antibodies. Application of this assay as a routine diagnostic tool requires long-term storage of FL-BP180, which can result in substantial loss of expression. Here, we show that the disaccharide trehalose, a natural cryo- and lyoprotectant, is capable of preserving the FL-BP180 antigen expressed in Sf21 insect cells under various (dry) storage conditions including 40degreesC, room temperature (RT), 4-8, -20, and -80 degreesC. The protective effect was dose-dependent reaching a maximum at about 200 mM trehalose. Trehalose was superior to other sugars or conventional cryoprotective agents (e.g. sucrose, myo-inositol, DMSO) in preventing greatly reduced antigen expression. Trehalose conserved the expression of both extra- and intracellular epitopes of FL-BP180. Interestingly, protection of the intracellular domain was only observed when trehalose was introduced into the cytosol. Trehalose significantly prolonged the storage time of FL-BP180 expressed in Sf21 insect cells, thus permitting the routine use of the IF assay in clinics for the detection of serum antibodies. The method described here has potential applications for the preservation of other transmembrane proteins. (C) 2003 Elsevier Science B.V. All rights reserved.
Reuss, O.R., Kurschner, M., Dilsky, S., Horbaschek, M., Schenk, W.A., Zimmermann, U., Sukhorukov, V.L.: Interaction of fluorinated lipophilic ions with the plasma membrane of mammalian cells studied by electrorotation and dielectrophoresis.JOURNAL OF ELECTROSTATICS.56,419-434 (2002).
Two complementary AC electrokinetic techniques electrorotation (ROT) and dielectrophoresis (DEP) were employed for studying the interaction of the fluorinated tungsten anion [W(CO)(5)(SC6H4CF3)](-) and a number of its derivatives with the plasma membrane of mouse myeloma cells. The use of microstructured electrodes allowed the measurements to be performed over wide ranges of field frequency and medium conductivity. At micromolar concentrations, most of the fluorinated anions gave rise to dramatic changes in the low-frequency part of both ROT and DEP spectra of cells, indicating that these compounds acted as lipophilic anions capable of introducing large amounts of mobile charges into the plasma membrane. Application of the theoretical models linking the ROT and DEP responses yielded not only the passive electrical properties of the plasma membrane and cytosol but also the ion transport parameters, such as the surface concentration and translocation rate of the lipophilic ions dissolved in the plasma membrane. This study demonstrated that combined ROT and DEP measurements can be employed as a powerful experimental tool for the analysis of the complex relationships between the molecular structure and membrane permeability of charged lipophilic compounds. Due to their strong lipophilicity and fairly low cytotoxicity, the fluorinated tungsten compounds presented here appear to provide a promising new class of field-sensitive molecular probes for membrane structure and transport studies. (C) 2002 Elsevier Science B.V. All rights reserved.
Shirakashi, R., Kostner, C.M., Muller, K.J., Kurschner, M., Zimmermann, U., Sukhorukov, V.L.: Intracellular delivery of trehalose into mammalian cells by electropermeabilization.JOURNAL OF MEMBRANE BIOLOGY.189,45-54 (2002).
The disaccharide trehalose is increasingly being used as a very efficient stabilizer of cells, membranes and macromolecules during cryo- and lyoconservation. Although extracellular trehalose can reduce cryo- and lyodamage to mammalian cells, the sugar is required on both sides of the plasma membrane for maximum protection efficiency. In the present study, mouse myeloma cells were loaded with the disaccharide by means of reversible electropermeabilization in isotonic trehalose-substituted medium, which contained 290 mm trehalose as the major solute. By using the membrane-impermeable fluorescent dye propidium iodide as the reporter molecule, optimum electropulsing conditions were found, at which most permeabilized cells survived and recovered (i.e., resealed) their original membrane integrity within a few minutes after electric treatment. Microscopic examination during the resealing phase revealed that electropulsed cells shrank gradually to about 60% of their original volume. The kinetics of the dye uptake and the volumetric response of cells to electropulsing were analyzed using a theoretical model that relates the observed cell volume changes to the solute transport across the transiently permeabilized cell membrane. From the best fit of the model to the experimental data, the intracellular trehalose concentration in electropulsed cells was estimated to be about 100 mm. This loading efficiency compares favorably to other methods currently used for intracellular trehalose delivery. The results presented here point toward application of the electropermeabilization technique for loading cells with membrane-impermeable bioprotectants, with far-reaching implications for cryo- and lyopreservation of rare and valuable mammalian cells and tissues.
Mussauer, H., Sukhorukov, V.L., Zimmermann, U.: Trehalose improves survival of electrotransfected mammalian cells.CYTOMETRY.45,161-169 (2001).
Background: Electropermeabilization is widely used for introduction of DNA and other foreign molecules into eukaryotic cells. However, conditions yielding the greatest molecule uptake and gene expression can result in low cell survival. In this study, we assessed the efficiency of trehalose for enhancing cell viability after excessive electropermeabilization. This disaccharide was chosen because of its capability of stabilizing cell membranes under various stressful conditions, such as dehydration and freezing. Materials and Methods: Various mammalian cell lines were electropermeabilized by single exponentially decaying electric Pulses of few kV/cm strength and of several-microsecond duration. Propidium iodide (PI) and a plasmid encoding green fluorescent protein (GFP), respectively, served as reporter molecules. The effects of trehalose on PI-uptake, GFP gene expression, transfection yield, and short- and long-term viability were analyzed by flow cytometry and electronic cell counting. Results: The substitution of inositol by trehalose in pulse media protected cells against field-induced cell lysis. The protection effect saturated at about 40 -50 mM trehalose. Transfection yield and gene expression were not significantly affected by trehalose. But the transfection efficiency was generally higher in the presence of trehalose, mainly because of the increased cell survival. Conclusions: We demonstrated that trehalose-substituted media are superior to standard trehalose-free pulse media for improving cell survival and achieving higher electrotransfection efficiency. Cytometry 45:161-169, 2001. (C) 2001 Wiley-Liss, Inc.
Muller, K.J., Sukhorukov, V.L., Zimmermann, U.: Reversible electropermeabilization of mammalian cells by high-intensity, ultra-short pulses of submicrosecond duration.JOURNAL OF MEMBRANE BIOLOGY.184,161-170 (2001).
Mouse myeloma cells were electropermeabilized by single square-wave electric pulses with amplitudes of up to similar to 150 kV/cm and durations of 10-100 nsec. The effects of the field intensity, pulse duration and medium conductivity on cell viability and field-induced uptake of molecules were analyzed by quantitative flow cytometry using the membrane-impermeable fluorescent dye propidium iodide as indicator molecule. Despite the extremely large field strengths, the majority of cells survived the exposure to ultra-short field pulses. The electrically induced dye uptake increased markedly with decreasing conductivity of the suspending medium. We assigned this phenomenon to the transient electrode-formation (stretching) force that assumes its maximum value if cells are suspended in low-conductivity media, i.e., if the external conductivity sigma (e) is smaller than that of the cytosol sigma (i). The stretching force vanishes when sigma (e) is equal to or larger than sigma (i). Due to their capability of delivering extremely large electric fields, the pulse power systems used here appear to be a promising tool for the electropermeabilization of very small cells and vesicles (including intracellular organelles, liposomes, etc.).
Kriegmaier, M., Zimmermann, M., Wolf, K., Zimmermann, U., Sukhorukov, V.L.: Dielectric spectroscopy of Schizosaccharomyces pombe using electrorotation and electroorientation.BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS.1568,135-146 (2001).
Two complementary AC electrokinetic techniques electrorotation (ER) and electroorientation (EO) enabled the dielectric characterization of the rod-shaped fission yeast Schizosaccharomyces pombe. The use of microstructured electrodes allowed both ER and EO measurements to be performed over wide ranges of field frequency and medium conductivity. Due to their layered structure, living S. pombe cells exhibited up to three well resolved peaks in their ER spectra and also two distinct orientations, i.e., parallel or perpendicular to the imposed linear Field. Heat treatment and enzymatic protoplast isolation led to dramatic changes in the electrokinetic behavior of fission yeast. Application of the theoretical models linking the ER and EO spectra yielded the dielectric parameters of the major structural units of S. pombe cells (cell wall, plasma membrane and cytosol). The dielectric characterization of yeasts has an enormous impact in biotechnology and biomedicine, because electric field pulse techniques (electrofusion and electropermeabilization) are widely used for production of transgenic yeast strains of economic importance. The present study also showed that combined ER and EO measurements can be employed as a powerful diagnostic tool for analyzing changes in yeast structure and physiology upon exposure to various stress conditions. (C) 2001 Elsevier Science B.V, All rights reserved.
Sukhorukov, V.L., Meedt, G., Kurschner, M., Zimmermann, U.: A single-shell model for biological cells extended to account for the dielectric anisotropy of the plasma membrane.JOURNAL OF ELECTROSTATICS.50,191-204 (2001).
For modeling the polarization of biological cells in electric fields and related AC electrokinetical phenomena, such as electrorotation, dielectrophoresis, etc., dielectric isotropy of the plasma membrane and other cellular compartments is usually assumed. However, this traditional assumption is no longer valid in the case of cell membranes containing mobile charges introduced by the adsorbed hydrophobic ions, such as dipicrylamine, tetraphenylborate, etc. Once partitioned into the membrane, the hydrophobic ions can move freely in the plane of the membrane thus increasing the tangential component of membrane conductivity sigma (mt). On the contrary, only finite charge displacement, i.e. translocation of hydrophobic ions between the two membrane boundaries, can be induced by the held component normal to the plasma membrane plane. This relaxational effect causes a dielectric dispersion (e.g. in the kHz-MHz range) with a marked increase of the radial membrane permittivity epsilon (mr) at low field frequencies. In this study we extended the single-shell spherical model of cells in order to account for the dielectrically anisotropic plasma membrane. In contrast to the usual approach, where the plasma membrane permittivity and conductivity are viewed as scalar quantities, these membrane parameters are treated as tensors in the anisotropic membrane model. Calculations based on the new model showed that the tangential conductivity of hydrophobic ions can induce noticeable changes in the low-frequency part of the electrokinetic spectra of cells. (C) 2001 Elsevier Science B.V. All rights reserved.
Sukhorukov, V.L., Kurschner, M., Dilsky, S., Lisec, T., Wagner, B., Schenk, W.A., Benz, R., Zimmermann, U.: Phloretin-induced changes of lipophilic ion transport across the plasma membrane of mammalian cells.BIOPHYSICAL JOURNAL.81,1006-1013 (2001).
The adsorption of the hydrophobic anion [W(CO)(5)CN](-) to human lymphoid Jurkat cells gave rise to an additional anti-field peak in the rotational spectra of single cells, indicating that the cell membrane displayed a strong dielectric dispersion in the kilohertz to megahertz frequency range. The surface concentration of the adsorbed anion and its translocation rate constant between the two membrane boundaries could be evaluated from the rotation spectra of cells by applying the previously proposed mobile charge model. Similar single-cell electrorotation experiments were performed to examine the effect of phloretin, a dipolar molecule known to influence the dipole potential of membranes, on the transport of [W(CO)SCN]- across the plasma membrane of mammalian cells. The adsorption of [W(CO)5CN]- was significantly reduced by phloretin, which is in reasonable agreement with the known phloretin-induced effects on artificial and biological membranes. The IC50 for the effect of phloretin on the transport parameters of the lipophilic ion was similar to 10 muM. The results of this study are consistent with the assumption that the binding of phloretin reduces the intrinsic dipole potential of the plasma membrane. The experimental approach developed here allows the quantification of intrinsic dipole potential changes within the plasma membrane of living cells.
Zimmermann, U., Friedrich, U., Mussauer, H., Gessner, P., Hamel, K., Sukhoruhov, V.: Electromanipulation of mammalian cells: Fundamentals and application.IEEE TRANSACTIONS ON PLASMA SCIENCE.28,72-82 (2000).
Electroinjection of membrane-impermeable xenomolecules into freely suspended mammalian tells (so-called electroporation) and cell-to-cell electrofusion are powerful tools for manipulation of the genom and the cytosol of tells, Both field pulse techniques are based on the temporary increase of the membrane permeability due to reversible electrical breakdown of the plasma membrane upon application of external high-intensity field pulses of very short duration. Membrane charging and permeabilization caused by high-intensity field pulses are preceded and accompanied by transient electrodeformation forces, which lead to an elongation of the cells in low-conductivity media, thus affecting the membrane area of electropermeabilization in response to a breakdown pulse, Transient stretching force assumes a maximum value in low-conductivity pulse media. This facilitates incorporation of membrane-impermeable xenomolecules and field-mediated hybridization as well. Therefore, high and reproducible yields of(genetically) manipulated cells can be expected provided that: 1) the duration of the high-intensity field pulses does not exceed about 100 mu s and 2) that the (pulse or fusion) media are hypo-osmolar and exhibit a relatively low conductivity. Such media are also beneficial because field-induced apoptosis does not occur under these conditions tin contrast to highly conductive media). Indeed, electroporation and electrofusion protocols that fulfill these requirements lead: I) to high incorporation rates of plasmids CDNA) or artificial chromosomes into living cells without deterioration and 2) to the production of hybridoma cells (by fusion of tumor-infiltrating lymphocytes with heteromyeloma cells), which secrete functional human monoclonal antibodies. Human monoclonal antibodies that bind to and induce apoptosis in autologous tumor cells are promising agents for cancer treatment, as shown by first clinical trials.
Kurschner, M., Nielsen, K., von Langen, J.R.G., Schenk, W.A., Zimmermann, U., Sukhorukov, V.L.: Effect of fluorine substitution on the interaction of lipophilic ions with the plasma membrane of mammalian cells.BIOPHYSICAL JOURNAL.79,1490-1497 (2000).
The effects of the anionic tungsten carbonyl complex [W(CO)(5)SC6H5](-) and its fluorinated analog [W(CO)(5)SC6F5](-) on the electrical properties of the plasma membrane of mouse myeloma cells were studied by the single-cell electrorotation technique. At micromolar concentrations, both compounds gave rise to an additional antifield peak in the rotational spectra of cells, indicating that the plasma membrane displayed a strong dielectric dispersion. This means that both tungsten derivatives act as lipophilic ions that are able to introduce large amounts of mobile charges into the plasma membrane. The analysis of the rotational spectra allowed the evaluation not only of the passive electric properties of the plasma membrane and cytoplasm, but also of the ion transport parameters, such as the surface concentration, partition coefficient, and translocation rate constant of the lipophilic anions dissolved in the plasma membrane. Comparison of the membrane transport parameters for the two anions showed that the fluorine-substituted analog was more lipophilic, but its translocation across the plasma membrane was slower by at least one order of magnitude than that of the parent hydrogenated anion.
Mussauer, H., Sukhorukov, V.L., Haase, A., Zimmermann, U.: Resistivity of red blood cells against high-intensity, short-duration electric field pulses induced by chelating agents.JOURNAL OF MEMBRANE BIOLOGY.170,121-133 (1999).
The interaction of human red blood cells (RBCs) with diethylenetriamine-pentaacetic acid (DTPA) or its Gd-complex (Magnevist, a widely used clinical magnetic resonance contrast agent containing free DTPA ligands) led to the following, obviously interrelated phenomena. (i) Both compounds protected erythrocytes against electrohemolysis in isotonic solutions caused by a high-intensity DC electric field pulse. (ii) The inhibition of electrohemolysis was observed only when cells were electropulsed in low-conductivity solutions. (iii) The uptake of Gd-DTPA by electropulsed RBCs was relatively low. (iv) (Gd-) DTPA reduced markedly deformability of erythrocytes, as revealed by the electrodeformation experiments using high-frequency electric fields. Taken together, the results indicate that (Gd-) DTPA produce stiffer erythrocytes that are more resistant to electric field exposure. The observed effects of the chelating agents on the mechanical properties and the electropermeabilization of RBCs must have an origin in molecular changes of the bilayer or membrane-coupled cytoskeleton, which, in turn, appear to result from an alteration of the ionic equilibrium (e.g., Ca2+ sequestration) in the Vicinity of the cell membrane.
Esch, M., Sukhorukov, V.L., Kurschner, M., Zimmermann, U.: Dielectric properties of alginate beads and bound water relaxation studied by electrorotation.BIOPOLYMERS.50,227-237 (1999).
The electrical and dielectric properties of Ba2+ and Ca2+ cross-linked alginate hydrogel beads were studied by means of single-particle electrorotation. The use of microstructured electrodes allowed the measurements to be performed over a wide range of medium conductivity from about 5 mS/m to 1 S/m. Within a conductivity range, the beads exhibited measurable electrorotation response at frequencies above 0.2 MHz with two well-resolved co- and antifield peaks. With increasing medium conductivity, both peaks shifted toward higher frequency and their magnitudes decreased greatly. The results were analyzed using various dielectric models that consider the beads as homogeneous spheres with conductive loss and allow the complex rotational behavior of beads to be explained in terms of conductivity and permittivity of the hydrogel. The rotation spectra could be fitted very accurately by assuming (a) a linear relationship between the internal hydrogel conductivity and the medium conductivity, and (b) a broad internal dispersion of the hydrogel centered between 20 and 40 MHz. We attribute this dispersion to the relaxation of water bound to the polysaccharide matrix of the beads. The dielectric characterization of alginate hydrogels is of enormous interest for biotechnology and medicine, where alginate beads are widely used for immobilization of cells and enzymes, for drug delivery, and as microcarriers for cell cultivation. (C) 1999 John Wiley & Sons, Inc.
Kurschner, M., Nielsen, K., Andersen, C., Sukhorukov, V.L., Schenk, W.A., Benz, R., Zimmermann, U.: Interaction of lipophilic ions with the plasma membrane of mammalian cells studied by electrorotation.BIOPHYSICAL JOURNAL.74,3031-3043 (1998).
The electrical properties of biological and artificial membranes were studied in the presence of a number of negatively charged tungsten carbonyl complexes, such as [W(CO)(5)(CN)](-), [W(CO)(5)(NCS)](-), [W-2(CO)(10)(CN)](-), and [W(CO)(5)(SCH2C6H5)](-), using the single-cell electrorotation and the charge-pulse relaxation techniques. Most of the negatively charged tungsten complexes were able to introduce mobile charges into the membranes, as judged from electrorotation spectra and relaxation experiments. This means that the tungsten derivatives act as lipophilic anions. They greatly contributed to the polarizability of the membranes and led to a marked dielectric dispersion (frequency dependence of the membrane capacitance and conductance). The increment and characteristic frequency of the dispersion reflect the structure, environment, and mobility of the charged probe molecule in electrorotation experiments with biological membranes. The partition coefficients and the translocation rate constants derived from the electrorotation spectra of cells agreed well with the corresponding data obtained from charge-pulse experiments on artificial lipid bilayers.
Sukhorukov, V.L., Benkert, R., Obermeyer, G., Bentrup, F.W., Zimmermann, U.: Electrorotation of isolated generative and vegetative cells, and of intact pollen grains of Lilium longiflorum.JOURNAL OF MEMBRANE BIOLOGY.161,21-32 (1998).
The dielectric structure of mature pollen of the angiosperm Lilium longiflorum was studied by means of single-cell electrorotation. The use of a microstructured four-electrode chamber allowed the measurements to be performed over a wide range of medium conductivity from 3 to 500 mS m(-1). The rotation spectra of hydrated pollen grains exhibited at least three well-resolved peaks in the kHz-MHz frequency range, which obviously arise due to the multilayered structure of pollen grains. The three-shell model can explain the complex rotational behavior of pollen grains in terms of conductivities, permittivities and thicknesses of the following compartments: the exine and intine of the pollen grain wall as well as the membrane and cytoplasm of the vegetative cell. However, the number of unknown parameters (more than 8) was too large to allow unambiguous values to be assigned to any of them. Therefore, to facilitate the evaluation of the pollen grain parameters, additional rotational measurements were made on isolated vegetative and generative cells. The rotation spectra of these cells could be fitted very accurately on the basis of the single-shell model by assuming a dispersion of the cytoplasm. The data on the membrane and cytoplasmic properties of isolated vegetative cells were then used for modeling the rotation spectra of pollen grains. This greatly facilitated the fitting of the theoretical model to the experimental data and allowed the dielectric properties of the major structural units to be determined. The dielectric characterization of pollen is of enormous interest for plant biotechnology, where pollen and isolated germ cells are successfully used for production of transgenic crop and drug plants of economic importance by means of electromanipulation techniques.
Sukhorukov, V.L., Mussauer, H., Zimmermann, U.: The effect of electrical deformation forces on the electropermeabilization of erythrocyte membranes in low- and high-conductivity media.JOURNAL OF MEMBRANE BIOLOGY.163,235-245 (1998).
Electrical breakdown of erythrocytes induces hemoglobin release which increases markedly with decreasing conductivity of the pulse medium. This effect presumably results from the transient, conductivity-dependent deformation forces (elongation or compression) on the cell caused by Maxwell stress. The deformation force is exerted on the plasma membrane of the cell, which can be viewed as a transient dipole induced by an applied DC electric field pulse. The induced dipole arises from the free charges that accumulate at the cell interfaces via the Maxwell-Wagner polarization mechanism. The polarization response of erythrocytes to a DC field pulse was estimated from the experimental data obtained by using two complementary frequency-domain techniques. The response is very rapid, due to the highly conductive cytosol. Measurements of the electrorotation and electrodeformation spectra over a wide conductivity range yielded the information and data required for the calculation of the deformation force as a function of frequency and external conductivity and for the calculation of the transient development of the deformation forces during the application of a DC-field pulse. These calculations showed that (i) electric force precedes and accompanies membrane charging (up to the breakdown voltage) and (ii) that under low-conductivity conditions, the electric stretching force contributes significantly to the enlargement of ``electroleaks'' in the plasma membrane generated by electric breakdown.
Nielsen, K., Schenk, W.A., Kriegmeier, M., Sukhorukov, V.L., Zimmermann, U.: Absorption of tungsten carbonyl anions into the lipid bilayer membrane of mouse myeloma cells.INORGANIC CHEMISTRY.35,5762-& (1996).
The anions of the tungsten carbonyl salts Na[W(CO)(5)(CN)]. 3H2(O) (1), K[W(CO)(5)(NCS)] (2), and K[W-2(CO)(10)(mu-SCN)] (3) are readily taken up by the lipid membrane of living cells. Cell rotation experiments show that at solution concentrations in the micromolar range the lipid bilayer absorbs up to 1.0 pmol cm(-2) of these anions, which leads to a pronounced increase of both the conductance, G(m), and the capacitance, C-m, of the membrane. 1-3 and similar salts are thus seen as ideal instruments to further develop models of mass transport through lipid membranes.
Djuzenova, C.S., Zimmermann, U., Frank, H., Sukhorukov, V.L., Richter, E., Fuhr, G.: Effect of medium conductivity and composition on the uptake of propidium iodide into electropermeabilized myeloma cells.BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES.1284,143-152 (1996).
The effects of ionic composition and conductivity of the medium on electropermeabilization of the plasma membrane of mammalian cells were studied. Temporal and spatial uptake of propidium iodide (PI) into field-treated cells was measured by means of flow cytometry, spectrofluorimetry and confocal laser scanning microscopy. Murine myeloma cells were electropulsed in iso-osmolar solutions. These contained 10-100 mu g ml(-1) PI at different conductivities (0.8-14 mS cm(-1)) and ionic strengths, adjusted by varying concentrations of K+, Na+, Cl- and SO42-. Field-induced incorporation of PI into reversibly permeabilized cells was (almost) independent of ionic composition and strength (at a fixed medium conductivity), but increased dramatically with decreasing medium conductivity at a fixed field strength. The time-course of PI uptake (which apparently reflected the resealing process of the membrane) could be fitted by a single-exponential curve (relaxation time about 2 min in the absence of Ca2+) and was independent of medium conductivity and composition. These and other data suggested that the expansion of the `electroleaks' during the breakdown process is field-controlled and depends, therefore, on the (conductivity-dependent) discharging process of the permeabilized membrane. The threshold field strength for dye uptake increased with increasing K+ concentration (about 0.6 kV cm(-1) in K+-free, NaCl-containing medium and about 0.9 kV cm(-1) in 30 mM KCl-containing medium). Also, the spatial uptake pattern of PI shifted from an asymmetric permeation through the cell hemisphere facing the anode to a more symmetric uptake through both hemispheres. These results suggested that the generated potential is superimposed on the (K+-dependent) resting membrane potential. Taking this into account, the breakdown voltage of the membrane was estimated to be about 1 V.
Sukhorukov, V.L., Zimmermann, U.: Electrorotation of erythrocytes treated with dipicrylamine: Mobile charges within the membrane show their `'signature'' in rotational spectra.JOURNAL OF MEMBRANE BIOLOGY.153,161-169 (1996).
In this study, electrorotation spectra of individual cells (that is, frequency dependence of cell rotation speed) have been proved to yield information not only about the passive electric properties of cell constituents, but also about the presence of mobile charges within the plasma membrane being part of ion carrier transport systems. Experiments on human erythrocytes pretreated with the lipophilic anion dipicrylamine (DPA) gave convincing evidence that these artificial mobile charges adsorbed to the plasma membrane contributed significantly to the rotational spectrum at relatively low conductivity of the external medium (2-5 mS m(-1)). Theoretical integration of the mobile charge concept into the single-shell model (viewing the cell as a homogenous sphere surrounded by a membrane) led to a set of equations which predicted electrorotational behavior of DPA-treated cells in dependence on medium conductivity. The quantitative data on the partition and the transmembrane translocation rate of the DPA anion extracted from the experimental rotational spectra agreed well with the corresponding literature values.
DJUZENOVA, C.S., SUKHORUKOV, V.L., KLOCK, G., ARNOLD, W.M., ZIMMERMANN, U.: EFFECT OF ELECTRIC-FIELD PULSES ON THE VIABILITY AND ON THE MEMBRANE-BOUND IMMUNOGLOBULINS OF LPS-ACTIVATED MURINE B-LYMPHOCYTES - CORRELATION WITH THE CELL-CYCLE.CYTOMETRY.15,35-45 (1994).
The effects of microsecond electropulses (1-5 kV/cm) on the viability of murine B lymphocytes and on their binding of antibodies by surface immunoglobulin (Ig) were studied in relation to the cell cycle. Before electropulsing, cultures given 48 h mitogenic stimulation showed at least two cell subpopulations, which were distinguishable by their levels of surface-Ig expression as assessed with FITC-labelled antibodies against mouse Ig. The immunofluorescence intensity of cells in S and G2/M phases was higher than that of G0/G1 cells. After exposure of the mitogen-stimulated lymphocytes to three exponentially decaying (time constant tau = 5-40 mu s) electric field pulses, dye exclusion assay showed that pulsing at 1 or 2 kV/cm (at 4 degrees C or 20 degrees C) did not cause permeabilization. Field strengths of 3, 4, or 5 kV/cm resulted in 20%, 45%, or 70% of dye-permeable cells, respectively, if the pulsed cells were transferred to phosphate-buffered saline on ice for 30 min. Incubation in full medium at 37 degrees C for 30 min (''resealing'') significantly decreased the percentage of permeabilized cells. Electropulsed G0/G1 cells were not only more resistant to direct electric exposure (tolerated higher field strengths) than S + G2/M cells but also responded better to resealing. The surface Ig of lymphocytes pulsed at higher fields and low temperature (4 or 5 kV/cm, tau = 5 mu s, three pulses, 4 degrees C) was less easily immunostained than in controls or in cells pulsed at 2 kV/cm or less. At 5 kV/cm those cells that were not permeabilized showed a greater reduction in immunostaining, especially if resealed. (C) 1994 Wiley-Liss, Inc.
SUKHORUKOV, V.L., DJUZENOVA, C.S., ARNOLD, W.M., ZIMMERMANN, U.: DNA, PROTEIN, AND PLASMA-MEMBRANE INCORPORATION BY ARRESTED MAMMALIAN-CELLS.JOURNAL OF MEMBRANE BIOLOGY.142,77-92 (1994).
Incorporation of DNA, protein, and plasma membrane during blockage by aphidicolin or by doxorubicin was studied by flow cytometry and electrororation of three cell lines (mouse-myeloma Sp2/0-Ag14, hybridoma H73C11, and fibroblast-like L929 cells). Drug-mediated arrest at the G1-S boundary (aphidicolin) or in G2/M (doxorubicin) did not arrest synthesis of either protein or total membrane area, the increases in which outstripped growth in cell volume and apparent cell area, respectively. Measurements of membrane capacity in normal and hypo-osmotic media showed that the drugs had not changed the fundamental bilayer, but that an increase in the number or size of microvilli must have occurred. Aphidicolin-arrested cells withstood hypo-osmotic stress better than untreated cells could, indicating that the membrane excess can be utilized as a reserve during rapid cell expansion. Hypo-osmotically treated cell populations exhibited only about half the coefficient of variance (CV) in membrane properties of cells at physiological osmolality. Populations of arrested cells exhibited the same high CV as asynchronous cells, indicating that chemical arrest does not give uniformly villated eel populations. However, the lowest CV values were given by some synchronized (aphidicolin-blocked, then released) populations. Removal of aphidicolin allowed most cells to progress through S and G2, and then divide. During these processes, the membrane excess was reduced. After removal of doxorubicin, the cells did not divide: some continued protein synthesis, grew abnormally large, and further increased their membrane excess. Membrane breakdown by electric pulsing (3 X 5kV/cm, 40 mu sec decay time) of aphidicolin-synchronized L cells in G2/M led to a 22% loss of plasma membrane (both the area-specific and the whole-cell capacitance were reduced), presumably via endocytosis-like vesiculation.
SUKHORUKOV, V.L., ARNOLD, W.M., ZIMMERMANN, U.: HYPOTONICALLY INDUCED CHANGES IN THE PLASMA-MEMBRANE OF CULTURED-MAMMALIAN-CELLS.JOURNAL OF MEMBRANE BIOLOGY.132,27-40 (1993).
Cells from three cell lines were electrorotated in media of osmotic strengths from 330 mOsm to 60 mOsm. From the field-frequency dependence of the rotation speed, the passive electrical properties of the surfaces were deduced. In all cases, the area-specific membrane capacitance (C(m)) decreased with osmolality. At 280 mOsm (iso-osmotic), SP2 (mouse myeloma) and G8 (hybridoma) cells had C(m) values of 1.01 +/- 0.04 muF/cm2 and 1.09 +/- 0.03 muF/cm2, respectively, whereas dispase-treated L-cells (sarcoma fibroblasts) exhibited C(m) = 2.18 +/- 0.10 muF/cm2. As the osmolality was reduced, the C(m) reached a well-defined minimum at 150 mOsm (SP2) or 180 mOsm (G8). Further reduction in osmolality gave a 7% increase in C(m), after which a plateau close to 0.80 muF/cm2 was reached. However, the whole-cell capacities increased about twofold from 200 mOsm to 60 mOsm. L-cells showed very little change in C(m) between 280 mOsm and 150 mOsm, but below 150 mOsm the C(m) decreased rapidly. The changes in C(m) correlate well with the swelling of the cells assessed by means of van't Hoff plots. The apparent membrane conductance (including the effect of surface conductance) decreased with C(m), but then increased again instead of exhibiting a plateau. The rotation speed of the cells increased as the osmolality was lowered, and eventually attained almost the theoretical value. All measurements indicate that hypo-osmotically stressed cells obtain the necessary membrane area by using material from microvilli. However, below about 200 mOsm the whole-cell capacities indicate the progressive incorporation of `'extra'' membrane into the cell surface.
POTAPENKO, A.Y., SAPAROV, S.M., AGAMALIEVA, M.A., LYSENKO, E.P., BEZDETNAYA, L.N., SUKHORUKOV, V.L.: FE-2+ IONS AND REDUCED GLUTATHIONE - CHEMICAL ACTIVATORS OF PSORALEN-SENSITIZED PHOTOHEMOLYSIS.JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY B-BIOLOGY.17,69-75 (1993).
The influence of Fe2+ ions and reduced glutathione (GSH) on the haemolysis of erythrocytes photosensitized (366 nm) by psoralen (PUVA haemolysis) was investigated. PUVA haemolysis was induced by the low fluence rate (24 W m-2) and high fluence (greater than 150 W m-2) of UV-A irradiation. It has been shown that Fe2+ ions and GSH activated PUVA haemolysis at both fluence rates of irradiation. PUVA haemolysis activation by Fe'' ions was more pronounced than that by GSH. It is supposed that activation caused by Fe2+ ions and GSH is connected with their ability to reduce lipid peroxides or psoralen peroxides with the subsequent formation of free radicals. The regeneration of endogenous Fe2+ by reduced glutathione is also possible.
WUNDERLICH, S., PLIQUETT, F., MORICH, E., KYAGOVA, A.A., SUKHORUKOV, V.L., POTAPENKO, A.Y.: PRE-IRRADIATED PHOTOOXIDIZED PSORALEN INDUCES HEMOLYSIS AND CHANGES IN ELECTRICAL PARAMETERS OF ERYTHROCYTE-MEMBRANES.BIOELECTROCHEMISTRY AND BIOENERGETICS.31,193-202 (1993).
The effects of pre-irradiated photo-oxidized psoralen (POP) on human erythrocytes (RBCs) were investigated. Psoralen in ethanol solutions was photo-oxidized and then added to a suspension of erythrocytes. The hemolysis and the electric impedance of the suspension were measured in the frequency range 5-20 MHz. The membrane capacity C(M), and the extracellular and intracellular conductivitieS kappa(a) and kappa(1) were calculated from these data. There appeared to be a threshold concentration of POP for hemolysis. When the concentration of POP slightly exceeded this threshold, the hemolytic effect rapidly increased to a maximum, but a further increase in POP concentration slowed down the rate of hemolysis. POP induced a decrease in extracellular conductivity and an increase in membrane capacity, but did not affect intracellular conductivity. The changes in extracellular conductivity correlated well with hemolysis.
POTAPENKO, A.Y., BEZDETNAYA, L.N., LYSENKO, E.P., AKHTYAMOV, S.N., TOMASHAEVA, S.K., SUKHORUKOV, V.L.: HYPOTHESIS OF THE INDUCTION OF PSORALEN PHOTOTOXIC EFFECTS THROUGH THE STAGE OF PHOTOOXIDIZED PSORALEN FORMATION - MODEL STUDIES OF ERYTHROCYTES.STUDIA BIOPHYSICA.124,205-223 (1988).
LYSENKO, E.P., POTAPENKO, A.Y., SUKHORUKOV, V.L.: DEPENDENCE OF ABSORPTION AND FLUORESCENCE EXCITATION-SPECTRA ON THE CONCENTRATION OF FUROCOUMARINS AND COUMARINS.BIOFIZIKA.33,747-750 (1988).
SUKHORUKOV, V.L., POTAPENKO, A.Y., CHUDINOVA, O.I., LYSENKO, E.P., BEZDETNAYA, L.N.: ENHANCING EFFECT OF CATECHOLAMINE DIHYDROXYPHENYLALANINE ON PSORALEN PHOTOSENSITIZED HEMOLYSIS.STUDIA BIOPHYSICA.124,235-238 (1988).
POTAPENKO, A.J., SUKHORUKOV, V.L., AGAMALIEVA, M.A., LYSENKO, E.P.: PHOTOHEMOLYSIS SENSITIZED BY PSORALEN, PARADOXICAL DEPENDENCE ON THE LIGHT-INTENSITY.DOKLADY AKADEMII NAUK SSSR.302,1258-1260 (1988).
EGOROV, S.Y., KRASNOVSKY, A.A., SUKHORUKOV, V.L., POTAPENKO, A.Y.: FUROCOUMARIN-PHOTOSENSITIZED FORMATION OF SINGLET MOLECULAR-OXYGEN IN AQUEOUS AND ALCOHOL SOLUTION.BIOFIZIKA.31,154-156 (1986).
KRASNOVSKY, A.A., SUKHORUKOV, V.L., EGOROV, S.Y., POTAPENKO, A.Y.: GENERATION AND QUENCHING OF SINGLET MOLECULAR-OXYGEN BY FUROCOUMARINS - DIRECT MEASUREMENTS.STUDIA BIOPHYSICA.114,149-158 (1986).
POTAPENKO, A.Y., BEZDETNAYA, L.N., LYSENKO, E.P., SUKHORUKOV, V.L., REMISOV, A.N., VLADIMIROV, Y.A.: MECHANISMS OF FUROCOUMARIN-SENSITIZED DAMAGE TO BIOLOGICAL-MEMBRANES.STUDIA BIOPHYSICA.114,159-170 (1986).
POTAPENKO, A.Y., WUNDERLICH, S., PLIQUETT, F., BEZDETNAYA, L.N., SUKHORUKOV, V.L.: PHOTOSENSITIZED MODIFICATION OF ERYTHROCYTE-MEMBRANES INDUCED BY FUROCOUMARINS.PHOTOBIOCHEMISTRY AND PHOTOBIOPHYSICS.10,175-180 (1986).
POTAPENKO, A.Y., BEZDETNAYA, L.N., WUNDERLICH, Z., SUKHORUKOV, V.L., PLIQUETT, F., DUBURG, G.Y.: EFFECTS OF ANTIOXIDANTS ON PHOTOOXIDATION OF PSORALEN.BIOFIZIKA.31,549-554 (1986).
SUKHORUKOV, V.L., POTAPENKO, A.Y.: OXIDATION OF LIPIDS BY DIMERIC PHOTOOXIDIZING 8-METHOXYPSORALENE.ZHURNAL FIZICHESKOI KHIMII.57,1320-1321 (1983).
SUKHORUKOV, V.L., POTAPENKO, A.Y., ZAKHAROVA, V.A.: OXIDATION OF UNSATURATED LIPIDS AND DIHYDROPHENYLALANINE IN THE ABSENCE OF LIGHT BY PHOTO-OXIDIZED FUROCOUMARINS.VOPROSY MEDITSINSKOI KHIMII.29,75-78 (1983).
KRASNOVSKII, A.A., SUKHORUKOV, V.L., POTAPENKO, A.Y.: PHOTOGENERATION OF SINGLET OXYGEN BY PSORALENS.BULLETIN OF EXPERIMENTAL BIOLOGY AND MEDICINE.96,1259-1261 (1983).
POTAPENKO, A.Y., SUKHORUKOV, V.L., DAVIDOV, B.V.: 8-METHOXYPSORALEN-SENSITIZED PHOTO-OXIDATION OF TOCOPHEROLS.PHOTOBIOCHEMISTRY AND PHOTOBIOPHYSICS.5,113-117 (1983).
MOSHNIN, M.V., POTAPENKO, A.Y., SUKHORUKOV, V.L., ZAKHAROVA, V.A.: DARK OXIDATION OF LIPOSOMES BY PHOTO-OXIDATION 8-METHOXYPSORALENE.ZHURNAL FIZICHESKOI KHIMII.57,2815-2817 (1983).
